A crucial and unanswered question in the field of food allergy research is why certain proteins elicit an IgE mediated immune response, while others are tolerated. One compelling hypothesis is that non-allergens are more digestible, resulting in sufficient protein degradation in the stomach and intestine to render the remaining fragments immunologically inert. Despite efforts to contrast the proteolytic stability of allergens and non-allergens, a clear link between digestibility and allergenicity has yet to be established. Confounding variables such as interactions with other components in the food matrix, cross-reactivity with other allergens, or the pathway of sensitization (e.g. alimentary canal versus respiratory tract) complicate the interpretation of experimental outcomes. In this project, we develop a highly defined system for exploring the relationship between digestibility and allergenicity. We hypothesize that the digestibility of a protein is dependent on its stability under acidic (pH <3.0) conditions. Using the major peanut allergen, Ara h 1, as a model system, we will computationally design acid-sensitive variants that are rapidly proteolyzed in gastric fluid. These mutants will provide optimal reagents for comparative studies relating pH-stability to digestibility and eventually to allergenicity. We will accomplish this goal through three aims: (1) benchmark pH-stability calculations on biophysical characterization of the Ara h 1 protein, (2) engineer mutations in Ara h 1 that specifically reduce stability at low, acid pH while preserving structure and function under neutral pH conditions and (3) relate pH-stability to digestibility using both established simulated gastric fluid assays and advanced dynamic digestion models. This project applies cutting-edge computational methods in molecular electrostatics to important issues in food allergy research. A detailed understanding of the molecular basis for food protein digestibility will help define its role in allergenicity, and allow us to develop more accurate protocols for predicting the allergenicity of new or genetically modified food proteins. Furthermore, the technologies proposed here may find future applications in increasing the safety of existing foods, such as the design of hypoallergenic peanuts. In order to achieve both short and long term goals, we have established a strong team of collaborators with expertise in computational structural biology, biophysical methods, protein expression and purification in a number of recombinant systems including plants, and immunological studies of the gut. This group has the necessary expertise and resources to ensure this ambitious and important project will succeed. Normally, proteins in food are completely broken down providing essential amino acids. However, incomplete digestion of some foods, such as peanuts, may induce an immune reaction against intact proteins absorbed in the intestine, resulting in the development of food allergies. We will genetically engineer a major peanut allergen such that it is highly susceptible to degradation in the strongly acidic juices of the stomach. These protein variants will help us better understand the role of digestion as a line of defense against allergens, and potentially lead to the development of hypoallergenic variants of peanuts and other foods.